Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative therapy for patients with hematologic malignancies. A significant obstacle to success following this therapeutic approach is the onset of Graft versus Host Disease (GVHD) syndromes, which pose a significant threat of morbidity, escalated and prolonged immunosuppressive therapy, organ dysfunction, impaired quality of life, and ultimately an increased risk for mortality.
Immunologic Response
The ability of an individual's immune responses to distinguish between his/her own antigens and foreign antigens is termed self-tolerance. The occasional breakdown of this self-tolerance can result in serious autoimmune diseases. Conversely, under certain conditions a foreign antigen acts as a tolerogen, establishing a state in which the animal fails to develop an immune reaction. Tolerance and immunity are alternative responses; it follows that tolerance is specific and is directed to particular antigenic determinants.
Graft-Versus-Host Disease (GVHD)
Immune tolerance of donor T cells to the host is broken in Graft-versus-host disease (GVHD), an immunological complication associated with high dose cancer treatment followed by allogeneic bone marrow and stem cell transplantation. Hess A D, Blood. 105(12): 4548-4549 (2005). Acute GVHD caused by mature donor lymphocyte alloreactivity to host tissue antigens is a major cause of morbidity and mortality following allogeneic blood and marrow transplantation (BMT). Multiple organs can be involved, including the skin, liver, and lungs, but the main cause of death appears to be damage to the intestinal tract (IT) small and large bowel, resulting in sepsis, diarrhea, and shock. Hill G R, Ferrara J L, Blood. 95(9): 2754-2759 (2000); Chen X, et al., Blood. 121(19): 3970-3980 (2013). A prominent finding is that GVHD disrupts IT microbial communities by inhibiting Paneth cell production of antimicrobial α-defensins. Eriguchi Y, et al., Blood. 120(1): 223-231 (2012).
A conceptual model for GVHD suggests that the disease is composed of phases that include tissue damage from conditioning therapy and activation of antigen-presenting cells, activation of donor T cells resulting in differentiation and migration, and finally an effector phase in which host tissue damage is mediated by inflammatory cytokines, such as TNFα and IL-1, and effector cells, most notably cytotoxic T cells. Pidala J, Cancer Control. 18(4): 268-276 (2011). It is additionally complicated by disturbances in pathways of immunological reconstitution and failure to acquire immunological tolerance, thereby resulting in both alloimmune and autoimmune attacks on multiple host tissues. Pavletic S Z, Fowler D H, Hematology Am Soc of Hematol Educ Program. 2012: 251-264 (2012).
Consistent with a 2005 National Institutes of Health (NIH) Consensus Conference, classification of GVHD is based on clinical presentation rather than time of onset. Pidala J, et al., Haematologica. 97(3): 451-458 (2012).
Acute GVHD manifestations include erythematosus or maculopapular rash, nausea and vomiting or diarrhea and cholestatic hepatitis, and historically were limited to within 100 days following HCT. Grading for acute GVHD divides acute GVHD into four stages based on the extent of involvement of the skin, liver and gastrointestinal tract. In stage I, there is a skin rash over <25% of the body, bilirubin is measured at 26-60 μmol/L, with a gut fluid loss of 500-1000 mL/day. In stage II, a skin rash covers 25-50% of the body, the bilirubin is measured at 61-137 μmol/L, and the gut loses from 1000-1500 mL/day. Stage III is characterized by involving >50% of the skin, the bilirubin is measured at 138-257 μmol/L, and the gut has lost more than 1500 mL/day. Stage IV is characterized by bullae desquamation (blisters with shedding of epidermal cells) of skin, the bilirubin exceeds >257 μmol/L, and the gut fluid loss is >2500 mL/day or ileus (disruption of the normal propulsive ability of the gastrointestinal tract; bowel obstruction).
Acute GVHD manifestations occurring more than 100 days after hematopoietic cell transplantation are classified as “persistent”, “recurrent”, or “late onset” acute GVHD, depending on the antecedent history of acute GVHD and absence of other chronic GVHD manifestations. Pidala J, et al., Haematologica. 97(3): 451-458 (2012).
Classic chronic GVHD, which can result in multiple clinical features involving multiple sites (eyes, gastrointestinal tract, liver, lungs, heart, bone marrow and kidneys), is defined by diagnostic manifestations of chronic GVHD without characteristic features of acute GVHD, with extensive skin involvement, elevated bilirubin, gastrointestinal tract involvement and progressive onset from acute GVHD as poor prognostic findings. Pidala J, et al., Haematologica. 97(3): 451-458 (2012).
An overlap subtype of GVHD, which displays features of both chronic and acute GVHD, is a condition with an adverse prognosis, functional impairment, and significantly higher symptom burden. Patients with acute features have significantly higher non-relapse mortality and lower overall survival rates. These patients suffer significant and diverse functional impairments compared to those with classic chronic GVHD, suggesting a systemic functional impairment beyond the more direct ramifications of concurrent acute GVHD manifestations. Pidala J, et al., Haematologica. 97(3): 451-458 (2012).
One of the major determinants for development and severity of acute GVHD in human transplantation is disparity in major and minor histocompatibility antigens, with an increasing number of mismatched antigens predicting greater risk of acute GVHD and nonrelapse mortality. Goulmy E, et al., N. Engl J Med. 334(5): 281-285 (1996); Lee S J, et al., Blood. 110(13): 4576-4583 (2007). Polymorphism in non-HLA genes, including cytokines such as tumor necrosis factor (TNF), interleukin 10 (IL-10), interferon gamma, KIR polymorphism, and NOD2/CARD15 gene polymorphism, also may contribute to the development and severity of acute GVHD. Pidala J, Cancer Control. 18(4): 268-276 (2011).
There are several hypotheses as to mechanisms of chronic GVHD pathogenesis: (1) thymic damage, in part mediated by prior acute GVHD, may impair the process of negative selection by thymic medullary epithelial cells that eliminate pathogenic T cells responsible for immunity; (2) the potential role of transforming growth factor-beta (TGF-β) has been supported by amelioration of chronic GVHD manifestations after neutralization of this cytokine in murine models, and the clinical observation of an inverse relationship between TGF-β signaling in CD4 and CD8 cells and the risk of chronic GVHD; and (3) B cells may play a role in chronic GVHD pathogenesis. Pidala J, Cancer Control. 18(4): 268-276 (2011).
Current Therapeutic Strategies
Approximately 50% of individuals that receive an allogeneic donor transplant will develop some degree of GVHD, and it is not clear that a major improvement has occurred in the ability to prevent or treat GVHD. Pavletic S Z, Fowler D H, Hematology Am Soc of Hematol Educ Program. 2012: 251-264 (2012).
Fatal GVHD, manifesting as chronic inflammatory destruction of the gut, lungs, skin, and other organs, can be completely abrogated in animals and humans by careful depletion of mature lymphocytes from the donor bone marrow graft prior to transplantation. However, when this approach has been taken in patients being treated for various cancers, the incidence of tumor relapse is greatly increased, due to the loss of graft vs. tumor effect (GVTE), which is characterized by an immune response to a graft recipient's tumor cells by a donor's transplanted immune cells in the bone marrow or peripheral blood. In fact, an inverse correlation exists between the severity of GVHD and the incidence of tumor relapse. Ringdén O, et al., Bone Marrow Transplant. 47(6): 831-837 (2012); Lee S J, et al., Blood. 100(2): 406-414 (2002); Signori A, et al., Bone Marrow Transplant. 47(11): 1474-1478 (2012).
Donor immune cells that have been implicated in the GVTE include CD4+ T cells, CD8+ T cells and natural killer (NK) cells. These cells are believed to use Fas-dependent killing and perforin degranulation to eradicate malignant cells. In addition to immune cells, cytokines such as interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been shown to potentiate GVTE. Ringdén O, et al., Br J Haematol. 147(5): 614-633 (2009).
Control of GVHD, with maintenance of GVTE, is the goal of current management, which relies heavily on steroid immunosuppression. Marshall S R, Nat Clin Pract Oncol. 3(6): 302-314 (2006). Glucocorticoids such as methylprednisolone or prednisone combined with cyclosporine are used to treat acute GVHD, but adverse effects with corticosteroids include increased risk of infections, hyperglycemia, psychosis, and myopathy. Prolonged use of corticosteroids can cause osteoporosis, cataract formation, and aseptic bone necrosis.
Clinical trials investigating GVHD unresponsive to steroid treatment have reported success with the following treatments: daclizumab, etanercept, extracorporeal photopheresis, infliximab, mycophenolate mofetil, pentostatin, rituximab, tacrolimus, thalidomide, and imatinib mesylate. Von Bonin M, et al., Cell Ther Transplant. 2(6): 10.3205/ctt-2010-en-000057.01 (2010); Wolff D, et al., Biol Blood Marrow Transplant. 17(1): 1-17 (2011). Other approaches, include antithymocyte globulin, denileukin diftitox, monoclonal antibodies (such as alemtuzumab), sirolimus, oral nonabsorbable corticosteroids such as budesonide or beclomethasone dipropionate, intra-arterial corticosteroids, and infusions of mesenchymal stem cells. Dignan F L, et al., Br J Haematol. 158(1):30-45 (2012); Deeg H J, Blood. 109(10): 4119-4126 (2007). None of these approaches has thus far proved satisfactory.
A solution for overcoming GVHD while preserving GVTE appears to be possible. In mice, several different strategies for preventing intestinal tract (IT) damage have been observed to reduce or eliminate GVHD mortality while preserving systemic alloreactivity and GVTE.
One strategy is to identify tumor specific antigens (TSAs) and the T cell clones recognizing them, so that these may be selectively expanded, while all other alloreactive clones are removed. Patterson A E, Korngold R, Biol Blood Marrow Transplant. 7(4): 187-196 (2001); Fanning S L, et al., J Immunol. 190(1): 447-457 (2013). The limited number of well-defined TSAs is an obstacle to this approach. So, too, is the removal of alloreactivity, which comprises a much broader, stronger, and less readily evaded response repertoire than that generated against a single TSA.
Four general strategies can be envisioned for protecting the gut against GVHD while preserving general alloreactivity: 1) Reduce accumulation of alloreactive effector lymphocytes at the most vulnerable IT sites via tighter endothelial barriers, decreased diapedesis and motility of alloreactive T cells (Teffs) (e.g., Th17 and Th1), and/or decreased gut-specific homing. 2) Inhibit IT neovascularization by donor derived endothelial cells (ECs) differentiating from precursors (EPCs) under hypoxic conditions—recently revealed to be a major source of IT pathology during GVHD. Komanduri K V, Blood. 121(17): 3303-3304 (2013); Leonhardt F, et al., Blood. 121(17): 3307-3318 (2013); Penack O, et al., Blood. 117(16): 4181-4189 (2011); Penack O, et al., J Natl Cancer Inst. 102(12): 894-908 (2010). 3) Activate and expand allospecific IT regulatory T cells (Tregs) to suppress the inflammatory and cytotoxic responses of effector T cells (Teffs) (e.g., Th17 and Th1) in a localized manner. In this scenario, Tregs protect the most vulnerable GVHD sites (gut mucosa), while alloreactive Teffs (e.g., Th17 and Th1) remain in circulation throughout the rest of the body, available to encounter and eliminate residual host derived tumor cells. 4) Reduce intestinal leakage of bacteria and bacterial products such as endotoxin, which induce local and systemic inflammation at sites of GVHD.
The potential utility of the first approach is supported by dramatically reduced GVHD in mice receiving allogenic cells from donors genetically defective for gut homing integrin α4β7 (Petrovic A, et al., Blood. 103(4): 1542-1547 (2004)), or retinoic acid receptors which transduce signals leading to α4β7 upregulation. Chen X, et al., Blood. 121(19): 3970-3980 (2013). Comparable protection against IT GVHD was seen when allograft donor lymphocytes were depleted of α4β7+ populations prior to transplantation. Petrovic A, et al., Blood. 103(4): 1542-1547 (2004). In these three cases where α4β7 mediated gut homing cells were absent, host syngeneic tumors were still strongly rejected. In transplant patients, maraviroc (a CCR5 blocker) prevented IT GVHD and acute (within 100 days) death, but was associated with ˜20% greater relapse at 1 year (vs. historical controls)—not statistically significant, but suggestive of immune suppression with respect to GVTE, perhaps due to the widespread distribution of CCR5 on immune cells. Reshef R, et al., N Engl J Med. 367(2): 135-145 (2012).
The potential utility of the second strategy (inhibition of neovascularization) is supported by analogous mouse studies with anti-vascular endothelial (VE)-cadherin mAb, which reduced IT neovascularization and IT GVHD while leaving anti-tumor alloreactivity intact. Penack O, et al., J Natl Cancer Inst. 102(12): 894-908 (2010).
The third approach also appears promising, based on clinical studies. Recently, an inverse correlation between Tregs bearing IT homing receptors α4β7 and acute GVHD in patients has been demonstrated. Engelhardt B G, et al., Bone Marrow Transplant. 46(3): 436-442 (2011); Engelhardt B G, et al., Exp Hematol. 40(12): 974-982 (2012). Addition of donor Tregs (not α4β7 selected) suppressed GVHD without significant early interference with anti-tumor immunity. Brunstein C G, et al., Blood. 117(3): 1061-1070 (2011).
The fourth approach has been supported by numerous pre-clinical and clinical studies showing reduced GVHD in mice and patients pre-treated with gut sterilization, or in some cases, pro-biotics prior to allogeneic transplantation, and by studies showing that inhibition of rho kinase prevents intestinal leak syndrome after irradiation. Mihaescu A, et al., Br J Surg. 98(1): 124-131 (2011).
Rho Associated Coiled-Coil Kinase (ROCK) Proteins
Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Rho-associated coiled-coil kinase (ROCK) proteins belong to the protein kinase A, G, and C family (AGC family) of classical serine/threonine protein kinases, a group that also includes other regulators of cell shape and motility, such as citron Rho-interacting kinase (CRIK), dystrophia myotonica protein kinase (DMPK), and the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs). The main function of ROCK signaling is regulation of the cytoskeleton through the phosphorylation of downstream substrates, leading to increased actin filament stabilization and generation of actin-myosin contractility. Morgan-Fisher M, et al., J Histochem Cytochem. 61(3):185-198 (2013).
Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases I and II (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK I (alternatively called ROK β) and ROCK II (also known as Rho kinase or ROK α) are 160-kDa proteins encoded by distinct genes. The mRNA of both kinases is ubiquitously expressed, but ROCK I protein is mainly found in organs such as liver, kidney, and lung, whereas ROCK II protein is mainly expressed in muscle and brain tissue. The two kinases have the same overall domain structure and have 64% overall identity in humans, with 89% identity in the catalytic kinase domain. Both kinases contain a coiled-coil region (55% identity) containing a Rho-binding domain (RBD) and a pleckstrin homology (PH) domain split by a C1 conserved region (80% identity) (See FIG. 1). Despite a high degree of homology between the two ROCKs, as well as the fact that they share several common substrates, studies have shown that the two ROCK isoforms also have distinct and non-redundant functions. For example, ROCK I has been shown to be essential for the formation of stress fibers and focal adhesions, whereas ROCK II is required for myosin II-dependent phagocytosis.
ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)-loaded Rho. Morgan-Fisher M, et al., J Histochem Cytochem. 61(3):185-198 (2013). Rho is a small GTPase which functions as a molecular switch, cycling between guanosine diphosphate (GDP) and guanosine triphosphate (GTP) bound states under signaling through growth factors or cell adhesion receptors. Morgan-Fisher M, et al., J Histochem Cytochem. 61(3):185-198 (2013). GTPases are hydrolase enzymes that bind and hydrolyze GTP. In a similar way to ATP, GTP can act as an energy carrier, but it also has an active role in signal transduction, particularly in the regulation of G protein activity. G proteins, including Rho GTPases, cycle between an inactive GDP-bound and an active GTP-bound conformation (See FIG. 2). The transition between the two conformational states occurs through two distinct mechanisms: activation by GTP loading and inactivation by GTP hydrolysis. GTP loading is a two-step process that requires the release of bound GDP and its replacement by a GTP molecule. Nucleotide release is a spontaneous but slow process that has to be catalyzed by RHO-specific guanine nucleotide exchange factors (RHOGEFs), which associate with RHO GTPases and trigger release of the nucleotide. The resulting nucleotide-free binary complex has no particular nucleotide specificity. However, the cellular concentration of GTP is markedly higher than that of GDP, which favors GTP loading, resulting in the activation of RHO GTPases.
Conversely, to turn off the switch, GTP has to be hydrolyzed. This is facilitated by RHO-specific GTPase-activating proteins (RHOGAPs), which stimulate the intrinsically slow hydrolytic activity of RHO proteins. Although guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are the canonical regulators of this cycle, several alternative mechanisms, such as post-translational modifications, may fine-tune the RHO switch. In addition, inactive RHO GTPases are extracted by RHO-specific guanine nucleotide dissociation inhibitors (RHOGDIs) from cell membranes to prevent their inappropriate activation and to protect them from misfolding and degradation. Garcia-Mata R, et al., Nat Rev Mol Cell Biol. 12(8): 493-504 (2011).
Many proteins aid in activating and inhibiting ROCK I and ROCK II. Table 1 shows molecules that regulate ROCK by direct binding. Morgan-Fisher M, et al., J Histochem Cytochem. 61(3):185-198 (2013). For example, small GTP-binding protein RhoA (which controls cell adhesion and motility through organization of the actin cytoskeleton and regulation of actomyosin contractility) (Yoshioka K, et al., Cancer Res. 59(8): 2004-2010 (1999)), RhoB (which is localized primarily on endosomes, has been shown to regulate cytokine trafficking and cell survival) and RhoC (which may be more important in cell locomotion) (Wheeler A P, Ridley A J, Exp Cell Res. 301(1): 43-49 (2004)), associate with and activate the ROCK proteins. Other GTP binding proteins, such as RhoE, Ras associated with diabetes (Rad) and Gem (a member of the RGK family of GTP-binding proteins within the Ras superfamily possessing a ras-like core and terminal extensions whose expression inhibited ROK beta-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase (see Ward Y, et al., J Cell Biol. 157(2): 291-302 (2002)), inhibit ROCK, binding at sites distinct from the canonical Ras binding domain (RBD). Association with the PDK1 kinase promotes ROCK I activity by blocking RhoE association.
TABLE 1Molecules that Regulate ROCK by Direct BindingBindingSite onPartnerROCKOutcome of InteractionCell TypesReferencesROCK IPDK1aa 375-415Retention of ROCK I at the(H) MalignantPinner andplasma membrane. Increasesmelanoma, (R)Sahal 2008cortical actin-myosinbreast cancer,contractility and increases(H) squamousamoeboid migration. Preventscell carcinomanegative regulation of ROCKIactivity by RhoE. PDK1 doesnot affect ROCK I kinaseactivity.MYBPHaa 17-535Reduces MLC(H) LungHosono et al.phosphorylation. Decreasesadenocarcinoma2011single-cell motility leading toreduced lung adenocarcinomainvasion and metastasis.RhoEaa 1-420Stress fiber disassembly and(H) SquamousRiento et al.suppresses hepatocellularcell and (H)2003; Pinnercarcinoma motility andhepatocellularand Sahalinvasiveness. In competitioncarcinoma, (H)2008; Ma Wwith PDK1 for the samemalignantet al. 2012binding site on ROCK I.melanomaRegulates ROCK I kinaseactivity.Shroom2aa 593-Shroom2 and ROCK interact(H, M)Dunlop et al.1062and regulate endothelial cellEndothelial cells2012; Farbercontractility. Reduced Shroomet al. 20112 mRNA levels have beenlinked to human colorectalcancer.ROCK IICoronin IBaa 1135-Inhibits ROCK II signaling to(H) BreastRana and1381myosinadenocarcinomaWorthylake2012CRMP-2Laa 1-543CRMP-2(L) inhibits ROCK II(H) Colon andYoneda et al.and -2Sactivity, resulting in alterationbreast2012of cell migration, actinadenocarcinoma,cytoskeleton organization, and(R) fibroblasts,decreased fibronectin matrix(Ca) kidneyassemblyepithelial cellsRaf1aa 1-543Reduces ROCK kinase(M) SkinEhrenreiter etactivity. Promotes STAT3/myccarcinoma, (M)al. 2005,activation and dedifferentiationprimary2009;in Ras-induced skin tumors.keratinocytes,Piazzolla etRegulates cell motility.(M) fibroblastsal., 2005;Niault et al.2009Dynamin 1aa 1135-Overexpression studies showed(R) Brain extractTurnuslime et1381that dynamin I is necessary foral. 2009appropriate ROCK II action onthe actin cytoskeleton inneuronal cells.MLCPaa 354-775ROCK II phosphorylates MBS(R) SmoothKimura et al.and inactivates MLCP.muscle cells1996; Wang etal. 2009Myosin IIaa 1152-Overexpression studies showed(P)Brain extract,Kawabata et1388myosin II to anchor ROCK II(M, R)al. 2004to stress fibersfibroblastsNPM/B23aa 5-553Enhances ROCK II activity.(M) FibroblastsMa Z et al.Leads to centrosome2006; Ferrettiamplification.et al. 2010P80aa 1-543Overexpression studies showed(R) Brain extractLeung et al.CRMP-1p80 CRMP-1 inhibits activity2002of recombinant ROCK IIkinase domain. ROCK IIphosphorylates p80 CRMP-1.ROCK I and IIGemaa 787-976Overexpression studies showed(H)Ward et al.(ROCK I),that Gem abolishes ROCK I-Neuroblastoma2002Full lengthdependent MLCROCK IIphosphorylation but not LIMKactivation. Prevents ROCK I-mediated cell rounding andneurite retraction inneuroblastoma cells. BindsROCK II.Radaa 787-976Overexpression studies showed(H)Ward et al.(ROCK I),that Rad binding preventsNeuroblastoma2002aa 807-976ROCK II-mediated cellROCK IIrounding and neurite retractionin neuroblastoma cells. BindsROCK I.Morgana/chpFull-lengthBinds and reduces ROCK II(H) EmbryonicFerretti et al.Ikinase activity. Inhibitskidney cells,2010ROCK II-NPM interaction.(M) embryonicBinds ROCK I containingfibroblastscomplexes.Shroom 3aa 726-926Recruitment of the ROCKs to(C, M)Nishimura and(ROCK I),apical junctions. IncreasesEmbryos, (Ca)Takeichi 2008aa 698-957MLC phosphorylation at apicalkidney epithelial(ROCK II)junctions. Shroom3-ROCKcellsinteraction is crucial forneuroepithelial cellarrangement and remodeling.CRMP, collapsing response mediator protein; LIMK, LIM domain kinase; MBS, myosin binding subunit; MLC, myosin light chain; MLCP, myosin light chain phosphatase; MYBPH, myosin binding protein H; NPM, nucleophosmin/B23; PDK1, phosphoinositide-dependent kinase I; ROCK, Rho-associated protein kinase.Canine (Ca), chick (C), human (H), mouse (M), porcine (P), or rat (R).
ROCK activation leads to a concerted series of events that promote force generation and morphological changes. These events contribute directly to a number of actin-myosin mediated processes, such as cell motility, adhesion, smooth muscle contraction, neurite retraction and phagocytosis. In addition, ROCK kinases play roles in proliferation, differentiation, apoptosis and oncogenic transformation, although these responses can be cell type-dependent. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008).
ROCK I and ROCK II promote actin-myosin mediated contractile force generation through the phosphorylation of numerous downstream target proteins, including ezrin/radixin/moesin (ERM), the LIM-kinases (LIMK), myosin light chain (MLC), and MLOC phosphatase (MLCP). ROCK phosphorylates LIM kinases-1 and -2 (LIMK1 and LIMK2) at conserved threonines in their activation loops, increasing LIMK activity and the subsequent phosphorylation of cofilin proteins, which blocks their F-actin-severing activity. ROCK also directly phosphorylates the myosin regulatory light chain, myosin light chain II (MLC), and the myosin binding subunit (MYPT1) of the MLC phosphatase to inhibit catalytic activity. Many of these effects are also amplified by ROCK-mediated phosphorylation and activation of the Zipper-interacting protein kinase (ZIPK), a serine/threonine kinase which is involved in the regulation of apoptosis, autophagy, transcription, translation, actin cytoskeleton reorganization, cell motility, smooth muscle contraction and mitosis, which phosphorylates many of the same substrates as ROCK (See FIG. 3).
The phosphorylation of MLC by ROCK provides the chemical energy for actin-myosin ratcheting, and also phosphorylates myosin light chain phosphatase (MLCP), thereby inactivating MLCP and preventing its dephosphorylation of MLC. Thus, ROCK promotes actin-myosin movement by activation and stabilization. Other known substrates of ROCK include the cytoskeleton related proteins such as the ERM proteins, and focal adhesion kinase (FAK). The ERM proteins function to connect transmembrane proteins to the cytoskeleton. Street C A, Bryan B A, Anticancer Res. 31(11): 3645-3657 (2011).
ROCK has been Linked to Apoptosis, Cell Survival, and Cell Cycle Progression
Rho-ROCK signaling has been implicated in cell cycle regulation. Rho-ROCK signaling increases cyclin D1 and Cip1 protein levels, which stimulate G1/S cell cycle progression. Morgan-Fisher M, et al., J Histochem Cytochem. 61(3):185-198 (2013). Polyploidization naturally occurs in megakaryocytes due to an incomplete mitosis, which is related to a partial defect in Rho-ROCK activation, and leads to an abnormal contractile ring lacking myosin IIA.
Rho-ROCK signaling also has been linked to apoptosis and cell survival. During apoptosis, ROCK I and ROCK II are altered to become constitutively-active kinases. Through proteolytic cleavage by caspases (ROCK I) or granzyme B (ROCK II), a carboxyl-terminal portion is removed that normally represses activity. Interaction with phosphatidyl inositol (3,4,5)-triphosphate (PIP3) provides an additional regulatory mechanism by localizing ROCK II to the plasma membrane where it can undertake spatially restricted activities, i.e. the regulation by localization of enzymatic activity. Phosphorylation at multiple specific sites by polo-like kinase 1 was found to promote ROCK II activation by RhoA. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008). Additional Serine/Threonine and Tyrosine kinases may also regulate ROCK activity given that more phosphorylations have been identified. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008). Specifically, protein oligomerization induces N-terminal trans-phosphorylation. Riento K, Ridley A J, Nat Rev Mol Cell Biol. 4(6): 446-456 (2003). Other direct activators include intracellular second messengers such as arachidonic acid and sphingosylphosphorylcholine which can activate ROCK independently of Rho. Furthermore, ROCK I activity can be induced during apoptosis. Mueller B K, et al., Nat Rev Drug Discov. 4(5): 387-398 (2005).
ROCK protein signaling reportedly acts in either a pro- or anti-apoptotic fashion depending on cell type, cell context and microenvironment. For instance, ROCK proteins are essential for multiple aspects of both the intrinsic and extrinsic apoptotic processes, including regulation of cytoskeletal-mediated cell contraction and membrane blebbing, nuclear membrane disintegration, modulation of Bc12-family member and caspase expression/activation and phagocytosis of the fragmented apoptotic bodies (FIG. 4). Mueller B K, et al., Nat Rev Drug Discov. 4(5): 387-398 (2005). In contrast, ROCK signaling also exhibits pro-survival roles (FIG. 4). Though a wealth of data exists to suggest both pro- and anti-survival roles for ROCK proteins, the molecular mechanisms that modulate these pleiotropic roles are largely unknown. Street C A, Bryan B A, Anticancer Res. 31(11): 3645-3657 (2011).
The importance of the cytoskeleton for various cellular functions, combined with the pleiotropy of ROCK targeted phosphorylation, accounts for the wide range of animal models in which ROCK inhibitors, such as Y-27632, have shown beneficial effects. These include experimental asthma, Alzheimer's disease, Parkinson's disease, systemic lupus erythematosis, cardiovascular disease, organ transplant, diabetes, and erectile dysfunction, among others. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008).
Data from ROCK I knockout mice supports their use to treat cardiovascular diseases. Using a variety of models that mimic chronic high blood pressure, partial or full deletion of ROCK I reduced cardiac fibrosis without affective cardiomyocyte hypertrophy. In addition, pressure overload was less effective at inducing cardiomyocyte apoptosis in ROCK I−/− mice relative to controls, suggesting a role for ROCK I in myocardial failure. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008).
Despite the considerable interest and the development of numerous potent ROCK inhibitors by different groups, there is little information in the literature reporting clinical trials with selective ROCK inhibitors. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008).
Selective ROCK inhibitors have not been extensively investigated in humans; only Fasudil® (also known as HA-1077) has been the subject of clinical trials. However, other ROCK inhibitors have been studied in the laboratory setting. Each ROCK inhibitor has different characteristics and specificity for the ROCK proteins. Examples of known ROCK inhibitors include, but are not limited to, Y-27632 2HCl (R&D Systems Inc., Minneapolis, Minn.), Triazovivin® (StemRD, Burlingame, Calif.), Slx-2119 (MedChem Express, Namiki Shoji Cop., LTD), WF-536 [(+)-®-4-(1-aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride] (Mitsubishi Pharma Corporation, Osaka, Japan), RK1-1447 (University of South Florida, Tampa, Fla., and Moffitt Cancer Center, Tampa, Fla.; Pireddu R, et al. “Pyridylthiazole-based ureas as inhibitors of Rho associated protein kinases (ROCK1 and 2).” Medchemcomm. 2012; 3(6):699-709.), Fasudil® (Asahi-KASEI Corp., Osaka, Japan), Fasudil® hydrochloride (R&D Systems Inc., Minneapolis, Minn.), GSK429286A (R&D Systems Inc., Minneapolis, Minn.), Rockout® (EMD Millipore, Philadelphia, Pa.), SR 3677 dihydrochloride (R&D Systems Inc., Minneapolis, Minn.); SB 772077B (R&D Systems Inc., Minneapolis, Minn.), AS 1892802 (R&D Systems Inc., Minneapolis, Minn.), H 1152 dihydrochloride (R&D Systems Inc., Minneapolis, Minn.), GSK 269962 (R&D Systems Inc., Minneapolis, Minn.), HA 1100 hydrochloride (R&D Systems Inc., Minneapolis, Minn.), and Glycyl-H-1152 dihydrochloride (R&D Systems Inc., Minneapolis, Minn.).
For example, GSK429286A is a selective inhibitor of both ROCK I and ROCK II with an IC50 of 14 nM and 63 nM, respectively. Rockout® is a cell-permeable indolopyridine compound that acts as a selective, reversible, and ATP-competitive inhibitor of ROCK with an IC50 of 25 μM; however, it does not inhibit the activation of ROCK (although it has been shown to affect cell migration, inhibit blebbing and decrease stress fibers). SR 3677 dihydrochloride is a selective ROCK inhibitor having IC50 values of 3 and 56 nM for ROCK II and ROCK I, respectively. SB 772077B dihydrochloride is a ROCK inhibitor with an IC50 value of 5.6 nM for ROCK I and ROCK II. AS 1892802 is an ATP-competitive ROCK inhibitor (IC50 values are 52 and 122 nM for human ROCK II and human ROCK I) and exhibits analgesic effects in rat models of inflammatory and noninflammatory arthritic pain. H 1152 dihydrochloride is a ROCK inhibitor that displays high selectivity over the other protein kinases (IC50 value for ROCK II is 0.012 μM). GSK 269962 is a ROCK inhibitor that exhibits IC50 values of 1.6 and 4 nM for ROCK I and ROCK II, respectively, and further displays greater than 30 fold selectivity for ROCK against a panel of serine/threonine kinases. Additionally, GSK 269962 has been shown to induce vasorelaxation in preconstricted rat aorta and lower blood pressure in a rat model of hypertension. HA 1100 hydrochloride is a cell-permeable active metabolite of Fasudil®, which produces ATP-competitive and reversible inhibition of ROCK and is about 100-fold selective over a range of other protein kinases (IC50 value of ROCK II is 575.44 nM and of ROCK I 758.58 nM (both human ROCK proteins)). Additionally, HA 1100 hydrochloride has been shown to inhibit neutrophil migration and produces potent vasodilatory effects in vivo. Glycyl-H-1152 dihydrochloride is a glycyl analog of the ROCK inhibitor H 1152 dihydrochloride that displays improved ROCK II selectivity (IC50 value is 0.0118 μM for ROCK II).
The isoquinoline derivative Fasudil® (and its monohydrochloride salt) is a cell-permeable Ca2+ antagonist that inhibits Rho-associated Kinase (ROCK II) having an IC50=1.6 μM. It was created as one of a series of compounds that inhibited PKA and PKC, but is significantly more potent for ROCK, with an IC50 at least 10-fold lower than for other kinases. However, its critical target has not been identified. Although ROCK is more potently inhibited by Fasudil® than related kinases such as PKA and PKC, and many effects of Fasudil® have been reproduced in model systems by structurally distinct inhibitors such as Y-27632, it has been hypothesized that the clinical effects of Fasudil® may actually result from the inhibition of other kinases or result from the combined inhibition of ROCK plus additional kinases, such as ZIPK or LIMK. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008). In animal models, Fasudil® has been shown to be effective in reversing blood vessel spasm and constriction that may occur after an episode of bleeding into the subarachnoid space surrounding the brain, a condition termed subarachnoid hemorrhage (SAH). Post-marketing surveillance studies on SAH patients have found that in over 1400 patients examined Fasudil® was well tolerated and safe. These findings have encouraged Fasudil® clinical trials for additional indications. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008).
Clinical trials with Fasudil® have focused on indications linked to the cardiovascular system. For example, human trials have been carried out to assess the efficacy of Fasudil® in: acute ischemic stroke, cerebral blood flow, stable angina pectoris, coronary artery spasm, heart failure-associated vascular resistance and constriction, pulmonary arterial hypertension essential hypertension, atherosclerosis and aortic stiffness. Clinical trials in the United States currently are underway to determine whether Fasudil® would be useful in treating atherosclerosis and hypercholesterolemia (ClinicalTrials.gov Identifier: NCT00120718), and Reynaud's phenomenon (ClinicalTrials.gov Identifier: NCT00498615), which is a vasospastic disorder that causes painful, pale and cold extremities. Olson M F, Curr Opin Cell Biol. 20(2): 242-248 (2008).
MLC phosphorylation is also important in the context of cell motility and signal transduction. Over the past 15 years, numerous cell functions have been revealed to be ROCK dependent, including leukocyte chemotaxis, diapedesis, cytokine secretion and responsiveness (Noma K, et al., J Clin Invest. 118(5): 1632-1644 (2008); Lämmermann T, et al., Nature. 453(7191): 51-55 (2008); Smith A, et al., J Cell Sci. 116(Pt 15): 3123-3133 (2003); Van Buul J D, Hordijk P L, Arterioscler Thromb Vasc Biol. 24(5): 824-833 (2004); Benais-Pont G, et al., J Cell Biol. 160(5): 729-740 (2003); Li B, et al., FEBS Lett. 580(17): 4252-4260 (2006); Nohria A, et al., Circ Res. 99(12): 1426-1432 (2006); Bardi G, et al., FEBS Lett. 542(1-3): 79-83 (2003); Lee J H, et al., J Cell Biol. 167(2): 327-337 (2004)), and tumor cell invasiveness and metastasis. Xue F, et al., Hepatol Res. 38(8): 810-817 (2008); Wong C C, et al., PLoS One. 3(7): e2779 (2008); Ogawa T, et al., Am J Transplant. 7(2): 347-355 (2007); Wang D S, et al., World J Gastroenterol. 10(2): 299-302 (2004); Vishnubhotla R, et al., Lab Invest. 87(11): 1149-1158 (2007); Kamal T, et al., Clin Cancer Res. 9(7): 2632-2641 (2003); Sahai E, Marshall C J, Nat Cell Biol. 4(6): 408-415 (2002); Croft D R, et al., Cancer Res. 64(24): 8994-9001 (2004); Mizukami Y, et al., J Biol Chem. 281(20): 13957-13963 (2006); Somlyo A V, et al., FASEB J. 17(2): 223-234 (2003); Somlyo A V, et al., Biochem Biophys Res Commun. 269(3): 652-659 (2000); Ying H, et al., Mol Cancer Ther. 5(9): 2158-2164 (2006); Bourguignon L Y, et al., Cell Motil Cytoskeleton. 43(4): 269-287 (1999). Additional targets of ROCK serine/threonine kinase activity include cytoskeletal ezrin, radixin, moesin, and focal adhesion kinase. Barreiro O, et al., J Cell Biol. 157(7): 1233-1245 (2002). Further, ROCK inhibits endothelial nitric oxide synthase (eNOS) production of NO, (Bivalacqua T J, et al., Proc Natl Acad Sci USA. 101(24): 9121-9126 (2004); Ming X F, et al., Mol Cell Biol. 22(24): 8467-8477 (2002)) thus, decreasing blood flow in NO regulated vascular beds. ROCK inhibition, therefore, increases blood flow in NO regulated vasculature, and also appears to enhance the integrity of endothelial barriers, decreasing capillary leakage and endothelial cell apoptosis. (Noma K, et al., J Clin Invest. 118(5): 1632-1644 (2008); Lämmermann T, et al., Nature. 453(7191): 51-55 (2008); Smith A, et al., J Cell Sci. 116(Pt 15): 3123-3133 (2003); Van Buul J D, Hordijk P L, Arterioscler Thromb Vasc Biol. 24(5): 824-833 (2004); Benais-Pont G, et al., J Cell Biol. 160(5): 729-740 (2003); Li B, et al., FEBS Lett. 580(17): 4252-4260 (2006); Nohria A, et al., Circ Res. 99(12): 1426-1432 (2006); Bardi G, et al., FEBS Lett. 542(1-3): 79-83 (2003); Lee J H, et al., J Cell Biol. 167(2): 327-337 (2004); Tominaga T, et al., J Cell Biol. 120(6): 1529-1537 (1993); Sánchez-Madrid F, del Pozo M A, EMBO J. 18(3): 501-511 (1999); Alevriadou B R, Am J Physiol Cell Physiol. 285(2): C250-252 (2003). Additionally, ROCK inhibition antagonizes vascular endothelial growth factor (VEGF) by more than one pathway. Etienne S, et al., J Immunol. 161(10): 5755-5761 (1998); Zhu F, et al., Med Oncol. 28(2): 565-571 (2011); Nakabayashi H, Shimizu K, Cancer Sci. 102(2): 393-399 (2011); Washida N, et al., Nephrol Dial Transplant. 26(9): 2770-2779 (2011); Kuno M, et al., Biochem Pharmacol. 77(2): 196-203 (2009); Takata K, et al., Mol Cancer Ther. 7(6): 1551-1561 (2008); Hata Y, et al., Jpn J Ophthalmol. 52(1): 16-23 (2008). Finally, recent findings indicate that, in the context of experimental autoimmune encephalomyelitis, ROCK inhibition promotes Treg differentiation by shifting macrophages from M1 to the Treg inducing M2 phenotype. Liu C, et al., PLoS OneE. 8(2): e54841 (2013).
A tenuous connection of cytoskeletal proteins to the pathology of GVHD has been suggested. Studies have reported that the presence of autoantibodies and alloantibodies to cytoskeletal proteins (microfilaments, microtubules and intermediate filaments) correlates with disease severity. Anti-cytoskeletal intermediate filament antibodies have been reported. Kapur R, et al., Haematologica. 93(11): 1702-1711 (2008). In patients with extensive cGVHD with generalized skin involvement and/or lung fibrosis, higher levels of anti-PDGFR antibodies have been detected; these antibodies were shown to activate the Ha-Ras, ERK1/2, ROS signal transduction cascade, leading to increased type I collagen-gene expression. Kapur R, et al., Haematologica. 93(11): 1702-1711 (2008).
Telmisartan
Telmisartan (Micardis®, Boehringer Ingelheim) is an FDA approved and licensed angiotensin receptor blocker (ARB). It has been safely used for over eighteen (18) years as an anti-hypertensive drug. Immune suppression has not been reported as an increased risk during the post-marketing period. The anti-hypertensive effects of telmisartan are now thought to reflect the combination of ARB activity, PPARγ activation, and ROCK inhibition. Telmisartan has been found to be an agonist for peroxisome proliferator activated receptor gamma (PPARγ). PPARγ, also known as the glitazone receptor, or NR1C3 (nuclear receptor subfamily 1, group C, member 3) is a member of a group of nuclear receptor protein that function as transcription factors, and play essential roles in the regulation of cellular differentiation, development, metabolism, and tumorigenesis. PPARγ agonists (e.g. glitazones) are used as insulin sensitizing drugs to treat type 2 diabetes, and also have anti-hyperlipidemia benefits. More recently, anti-inflammatory functions of PPARγ agonists have been elucidated, some of which appear to be due to reduction in the activity of ROCK. Telmisartan has been found to be as potent an inhibitor of ROCK as Y-27632, a specific ROCK inhibitor. Kobayashi N, et al., Am J Hypertens. 21(5): 576-581 (2008).
Because recognition of telmisartan as a PPARγ agonist has been slow, and its potency as a ROCK inhibitor is not widely appreciated, its potential as a GVHD attenuator has not been explored. Additionally, there may be concerns that any positive effects will not be cleanly attributable to a single mechanism of action. However, protection from GVHD by the ROCK inhibitor, Fasudil®, similar in potency and mechanism of action to Y-27632, has been demonstrated. Iyengar S, et al., Biol Blood Marrow Transplant. 20(8): 1104-1111 (2014). Fasudil® and Y-27632 have been much more extensively studied than telmisartan, over the past two decades, with respect to the three potentially protective mechanisms envisioned. Fasudil® occupies the ATP binding pocket of ROCK's enzymatically functional kinase domain, thereby preventing phosphorylation of myosin light chain II (MLC) and MLC phosphatase (MLCP). Riento K, Ridley A J, Nat Rev Mol Cell Biol. 4(6): 446-456 (2003); Yoneda A, et al., J Cell Biol. 170(3): 443-453 (2005); Wang Y, et al., Circ Res. 104(4): 531-540 (2009). Phosphorylation of MLC activates it for actin filament binding and ratcheting, while phosphorylation of MLCP prevents this enzyme from de-phosphorylating (inactivating) MLC. Thus, ROCK potentiates smooth muscle contraction from two angles, both inhibited by Fasudil®. This explains Fasudil®'s anti-spasmodic properties on arterial smooth muscle, and its anti-hypertensive effects in pre-clinical and clinical studies (Vicari R M, et al., J Am Coll Cardiol. 46(10): 1803-1811 (2005); Shimokawa H, et al., J Cardiovasc Pharmacol. 40(5): 751-761 (2002); Fukumoto Y, et al., J Cardiovasc Pharmacol. 49(3): 117-121 (2007); Otsuka T, et al., Circ J. 70(4): 402-408 (2006); Masumoto A, et al., Circulation. 105(13): 1545-1547 (2002); Mohri M, et al., J Am Coll Cardiol. 41(1): 15-19 (2003); Inokuchi K, et al., J Cardiovasc Pharmacol. 44(3): 275-277 (2004); Kishi T, et al., Circulation. 111(21): 2741-2747 (2005)), as well as anti-asthmatic effects in OVA induced asthma models. Witzenrath M, et al., Exp Toxicol Pathol. 60(1): 9-15 (2008). Fasudil®, unavailable for clinical use in Europe or USA, has been safely used in Japan and other Asian countries for almost two decades without evidence of immune suppression.
Additionally, rosiglitazone, a specific PPARγ agonist, has been shown to suppress GVHD inflammation in a similar mouse model although survival curves were not followed. Song E K, et al., Transpl Immunol. 27(2-3): 128-137 (2012). Telmisartan has been shown to have a protective effect against rat colitis, a condition that shares common pathways with GVHD. Arab H H, et al., PLoS One. 9(5): e97193 (2014).
Telmisartin has been shown to abrogate lymphocyte chemotaxis, in part by abrogation of SDF-1 induced chemotaxis. Walcher D, et al., Hypertension. 51(2): 259-266 (2008). Additionally, telmisartan may maintain gut endothelial barriers by protecting endothelial cells (ECs) from inflammation mediated destruction. Cianchetti S, et al., Atherosclerosis. 198(1): 22-28 (2008); Siragusa M, Sessa W C, Arterioscler Thromb Vasc Biol. 33(8): 1852-1860 (2013). Telmisartan has also been shown to prevent neovascularization in corneal systems. Usui T, et al., Invest Ophthalmol Vis Sci. 49(10): 4370-4376 (2008); Nagai N, et al., Invest Ophthalmol Vis Sci. 46(3): 1078-1084 (2005). Finally, telmisartan has been shown to increase the ratio of protective Tregs:autoreactive Th17 cells (Liu Z, et al., Atherosclerosis. 233(1): 291-299 (2014)), and a very recent report demonstrates anti-inflammatory effects of telmisartan in the setting of chemically induced acute colitis. Arab H H, et al., PLoS One. 9(5): e97193 (2014). Multiple genetic pathways activated by inflammation and oxidative stress, along with inflammatory cell infiltrates and gross pathology of weight loss and diarrhea, were attenuated by pre-treatment with telmisartan.
A recent study has shown that telmisartan may inhibit cell proliferation in colon cancer cells induced by disrupting nuclear translocation of C-terminal fragments of proheparin-binding epidermal growth factor like growth factor. Ozeki K, et al., PLoS One. 8(2): e56770 (2013).
Clinical Experience with Micardis® Brand of Telmisartan
The antihypertensive effects of Micardis® brand telmisartan have been demonstrated in multiple placebo-controlled clinical trials, studying a range of 20 to 160 mg; one of these examined the antihypertensive effects of telmisartan and hydrochlorothiazide in combination. Micardis® package insert. Ingelheim, Germany: Boehringer Ingelheim Int'l.; 2014. The studies involved a total of 1773 patients with mild to moderate hypertension (diastolic blood pressure of 95 to 114 mm Hg), 1031 of which were treated with telmisartan. Following once daily administration of telmisartan, the magnitude of blood pressure reduction from baseline after placebo subtraction was approximately (SBP/DBP) 6-8/6 mm Hg for 20 mg, 9-13/6-8 mm Hg for 40 mg, and 12-13/7-8 mm Hg for 80 mg telmisartan doses. Larger doses (up to 160 mg) did not appear to cause a further decrease in blood pressure.
The incidence of symptomatic orthostasis after the first dose in all controlled trials was low (0.04%). Upon initiation of antihypertensive treatment with telmisartan, blood pressure was reduced after the first dose, with a maximal reduction achieved by about four (4) weeks. Most of the blood pressure lowering effect was observed within the first two (2) weeks of treatment. With cessation of treatment with Micardis® tablets, blood pressure gradually returned to baseline values over a period of several days to one (1) week. The antihypertensive effect of telmisartan is not influenced by patient age, gender, weight, or body mass index. Blood pressure response in African American patients (usually a low-renin population) is noticeably less than that in Caucasian patients.
Drug Interactions and Cautions for Telmisartan
Telmisartan is contraindicated during pregnancy, and no subjects should be enrolled if there is a chance of pregnancy during the telmisartan treatment phase of a clinical trial.
In patients with an activated renin-angiotensin system, such as volume- or salt-depleted patients (e.g., those being treated with high doses of diuretics), symptomatic hypotension may occur after initiation of therapy with telmisartan. Patients should be taken off their non-telmisartan anti-hypertensives for two (2) days prior to administration of telmisartan. Blood chemistries, blood pressure, and urination frequency should be monitored to ensure adequate hydration and normokalemia prior to starting telmisartan.
Hyperkalemia may occur in patients on telmisartan, particularly in patients with advanced renal impairment, heart failure, on renal replacement therapy, or on potassium supplements, potassium-sparing diuretics, potassium-containing salt substitutes or other drugs that increase potassium levels.
As the majority of telmisartan is eliminated by biliary excretion, patients with biliary obstructive disorders or hepatic insufficiency can be expected to have reduced clearance.
As a consequence of inhibiting the renin-angiotensin-aldosterone system, changes in renal function may occur in susceptible individuals. In patients whose renal function may depend on the activity of the renin-angiotensin-aldosterone system (e.g., patients with severe congestive heart failure or renal dysfunction), treatment with angiotensin receptor antagonists has been associated with oliguria and/or progressive azotemia and (rarely) with acute renal failure and/or death.
In patients who are elderly, volume-depleted (including those on diuretic therapy), or with compromised renal function, co-administration of NSAIDs, including selective COX-2 inhibitors, with telmisartan, may result in deterioration of renal function, including possible acute renal failure. These effects are usually reversible.
Drugs without known telmisartan interactions include: acetaminophen, amlodipine, glyburide, simvastatin, hydrochlorothiazide, warfarin, and ibuprofen.
Telmisartan is not metabolized by the cytochrome P450 system and has no effects in vitro on cytochrome P450 enzymes, except for some inhibition of CYP2C19. Telmisartan is not expected to interact with drugs that inhibit cytochrome P450 enzymes; it is also not expected to interact with drugs metabolized by cytochrome P450 enzymes, except for possible inhibition of the metabolism of drugs metabolized by CYP2C19.
Adverse Events for Telmisartan
The most common adverse events (≥1%) reported in hypertension trials of Micardis® are back pain, sinusitis, and diarrhea (see Table 2). When Micardis® was used for the reduction of cardiovascular risk, the serious adverse events (≥1%) were intermittent claudication and skin ulcer. The incidence of adverse events was not dose-related and did not correlate with gender, age, or race of patients.
TABLE 2Adverse Events Occurring at an Incidence of ≥1% in Patients Treated with MICARDIS ® and at a Greater Rate Than Patients Treated with PlaceboTelmisartan n = 1455 (%)Placebo n = 380 (%)Upper respiratory 76tract infectionBack pain31Sinusitis32Diarrhea32Pharyngitis10
In addition to these adverse events, the following events occurred at a rate of ≥1% but were at least as frequent in the placebo group: influenza-like symptoms, dyspepsia, myalgia, urinary tract infection, abdominal pain, headache, dizziness, pain, fatigue, coughing, hypertension, chest pain, nausea, and peripheral edema. Discontinuation of therapy because of adverse events was required in 2.8% of 1455 patients treated with Micardis® tablets and 6.1% of 380 placebo patients in placebo-controlled clinical trials. The incidence of cough occurring with telmisartan in 6 placebo-controlled trials was identical to that noted for placebo-treated patients (1.6%).
Pharmacokinetics of Telmisartan
Following oral administration, peak concentrations (Cmax) of telmisartan are reached in 0.5 to 1 hour after dosing. Food slightly reduces the bioavailability of telmisartan, with a reduction in the area under the plasma concentration-time curve (AUC) of about 6% with the 40 mg tablet and about 20% after a 160 mg dose. The absolute bioavailability of telmisartan is dose dependent. At 40 and 160 mg the bioavailability is 42% and 58%, respectively. The pharmacokinetics of orally administered telmisartan are nonlinear over the dose range 20 to 160 mg, with greater than proportional increases of plasma concentrations (Cmax and AUC) with increasing doses. Telmisartan shows bi-exponential decay kinetics with a terminal elimination half-life of approximately 24 hours. Trough plasma concentrations of telmisartan with once daily dosing are about 10 to 25% of peak plasma concentrations. Telmisartan has an accumulation index in plasma of 1.5 to 2.0 upon repeated once daily dosing.
Telmisartan is highly bound to plasma proteins (>99.5%), mainly albumin and orosomucoid. Plasma protein binding is constant over the concentration range achieved with recommended doses. The volume of distribution for the highly lipophilic telmisartan is approximately 500 liters indicating additional tissue binding.
Following either intravenous or oral administration of 14C-labeled telmisartan, most of the administered dose (>97%) is eliminated unchanged in feces via biliary excretion; only minute amounts are found in urine (0.91% and 0.49% of total radioactivity, respectively).
To date, no ideal treatment or therapy exists that is effective to modulate GVHD pathology while preserving GVTE, following allogenic BMT.
The described invention provides methods for treating and preventing GVHD, increasing subject survival and preserving alloreactivity of transplanted T cells in transplant patients comprising administering a therapeutic amount of telmisartan, wherein the therapeutic amount may be effective to reduce the incidence of GVHD and to preserve GVTE in a patient receiving a transplant.